It has been demonstrated that the genes involved in initiating the synthesis of DNA, RNA and protein in bacteria are contained in one single structural unit named the macromolecular synthesis operon (MMS). The genes are part of a single transcription unit and have been identified as rpsU encoding ribosomal protein S21 involved in initiating translation, dnaG encoding the protein primase which initiates DNA replication and rpoD which encodes sigma-70 involved in initiating transcription. The operon structure is found in both gram negative bacteria, such as Escherichia coli and Salmonella typhimurium, and in gram positive bacteria such as Bacillus subtilis. The individual structural genes are conserved and have large areas of homology. On the other hand, the intergenic sequences between the structural gene within the operon are unique to each bacterial species. The MMS operon appears to be a central information processing unit for directing the flow of genetic information. The organization of the operon suggests that under certain physiological conditions there is a need for coordination of synthesis of the information macromolecules (DNA, RNA and protein) in the cell and hence a coregulation of the initiator genes. Since the synthesis of each class of macromolecule appears to be regulated at its initiation step, regulation of the MMS operon most likely plays a role in regulating cell growth.
The MMS operon contains three structural genes. The rpsU gene encodes the ribosomal protein S21 which is required for specific initiation of messenger RNA (mRNA) translation. The protein S21 interacts with a stretch of ribosomal RNA (rRNA) complementary to the mRNA ribosomal binding site called the Shine-Dalgarno sequence located at the 3' end of the 16S rRNA. Colicin E3 removes 50 nucleotides from the 3' terminus of 16S rRNA. E3 treated ribosomes cannot carry out polypeptide chain initiation nor chain elongation. In reconstitution experiments, E3 treated ribosomes bind all 30S proteins except S21. RNA protein cross-linking experiments demonstrate that protein S21 is cross-linked to the 3' dodecanucleotide of the 16S rRNA. The base-pairing potential of the 3' terminus of 16S rRNA depends on the functional state of the 30S subunit and the presence of S21, which is required for specific initiation of E. coli and phage MS2 mRNA translation.
Initiation of DNA replication requires a priming RNA which is synthesized by the dnaG gene product, primase. This protein binds to the phage G4 origin of replication. Primase also is known to interact with the multienzyme complex primosome to initiate synthesis of Okazaki fragments on the chromosomal replication fork-lagging strand of E. coli. Primase is the sole priming enzyme required for initiation of DNA replication at the origin of the E. coli chromosome. A parB mutation in the dnaG gene results in abnormal partition of chromosomes and was originally isolated as a thermosensitive mutant affecting DNA synthesis and cellular division. Thus, in addition to initiation of DNA replication, the dnaG gene appears to play some role in regulating cell division.
The ropD gene product sigma-70 is involved in the recognition of promoter sequences for the specific initiation of RNA transcription. Sigma-70 interacts with the core polymerase .alpha..sub.2 .beta..beta." conferring specificity for promoter sequences. Sigma-70 is a member of a large family of RNA polymerase sigma factors. Thus, the macromolecular synthesis operon gene products share a common mechanism. Through protein-nucleic acid interactions the gene products of the MMS operon bind specific nucleotide sequences. For example S21 binds the Shine-Dalgarno sequence/ribosome binding site, primase binds the origin of replication, and sigma-70 binds a promoter sequence. These interactions result in initiation of synthesis of protein, DNA or RNA respectively.
Antisense RNAs have been utilized both in nature and experimentally to regulate gene expression. For example antisense RNA is important in plasmid DNA copy number control, in development of bacteriophage P22. Antisense RNAs have been used experimentally to specifically inhibit in vitro translation of mRNA coding from Drosophila hsp23, to inhibit Rous sarcoma virus replication and to inhibit 3T3 cell proliferation when directed toward the oncogene c-fos. Furthermore, it is not necessary to use the entire antisense mRNA since a short antisense oligonucleotide can inhibit gene expression. This is seen in the inhibition of chloramphenicol acetyltransferase gene expression and in the inhibition of specific antiviral activity to vesicular stomatitus virus by inhibiting the N protein initiation site. Antisense oligonucleotides to the c-myc onocogene have been demonstrated to inhibit entry into the S phase but not the progress from G.sub.0 to G.sub.1. Finally, inhibition of cellular proliferation has been demonstrated by the use of antisense oligodeoxynucleotides to PCNA cyclin.
Antibiotics are important pharmaceuticals for the treatment of infectious diseases in a variety of animals including man. The tremendous utility and efficacy of antibiotics results from the interruption of bacterial (prokaryotic) cell growth with minimal damage or side effects to the eukaryotic host harboring the pathogenic organisms. All antibiotics destroy bacteria by interfering with the normal flow of genetic information. This is performed by inhibition of any one of the following: DNA replication, that is, DNA to DNA (for example, the drugs Novobiocin and Nalidixic acid); transcription, that is, DNA to RNA (for example, Rifampin); translation, that is, RNA to protein (for example, tetracyclines, erythromycin and kanamycin); or cell wall synthesis (for example, penicillins).
The present invention provides a new class of antibiotics and a method for the treatment of bacterial infections either generally or specifically. The antibiotics are antisense oligonucleotide sequences which bind mRNA transcribed from the MMS operon. This is a new method of treating bacterial infections by interfering with the fundamental structural unit that regulates the growth and replication of bacteria.